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OPEN An updated investigation on the dromedary camel (Camelus dromedarius) with special insight into the distribution of calcium‑binding proteins Abdelraheim H. Attaai1,3, Ahmed E. Noreldin2, Fatma M. Abdel‑maksoud1* & Manal T. Hussein1,3

Studying the cerebella of diferent animals is important to expand the knowledge about the cerebellum. Studying the camel cerebellum was neglected even though the recent research in the middle east and Asia. Therefore, the present study was designed to achieve a detailed description of the morphology and the cellular organization of the camel cerebellum. Because of the high importance of the calcium ions as a necessary moderator the current work also aimed to investigate the distribution of calcium binding proteins (CaBP) such as calbindin D-28K (CB), (PV) and calretinin (CR) in diferent cerebellar cells including the non-traditional . The architecture of camel cerebellum, as diferent mammals, consists of the medulla and three layered-cortex. According to our observation the cells in the granular layer were not crowded and many spaces were observed. CB expression was the highest by Purkinje cells including their dendritic arborization. In addition to its expression by the inhibitory (basket, stellate and Golgi neurons), it is also expressed by the excitatory granule cells. PV was expressed by Purkinje cells, including their primary arborization, and by the molecular layer cells. CR immunoreactivity (-ir) was obvious in almost all cell layers with varying degrees, however a weak or any expression by the Purkinje cells. The molecular layer cells and the Golgi and the non traditional large neurons of the granular layer showed the strongest CR-ir. Granule neurons showed moderate immunoreactivity for CB and CR. In conclusion, the results of the current study achieved a complete map for the neurochemical organization of CaBP expression and distribution by diferent cells in the camel cerebellum.

Te cerebellum plays a fundamental role for diverse motor functions, postural control, balance, and motor ­coordination1,2. It is also involved in cognitive functions such as language, learning, memory, and some emo- tional behaviors such as ­fear3,4. Te cerebellum consists of 2 main components, the cerebellar cortex and the medulla. Tere are several cellular components in the cerebellar cortex; the most distinguished morphologically are Purkinje cells (PCs), granule, Golgi and basket cells­ 5. Tis complex neural network gives rise to a massive signal-processing capability that plays a role in motor learning­ 6. Te inputs to the cerebellum come from the climbing fbers and the mossy fbers. Most of the neuronal subtypes in the cerebellar cortex are interneurons which synapse with PCs. Te later’s axons constitute the outputs, of the integrated signals, which rely on either small lying in the interior of the cerebellum or transmit information to structures outside the cerebellum.

1Department of Anatomy and Histology, Faculty of Veterinary Medicine, Assiut University, 71526 Assiut, Egypt. 2Department of Histology and Cytology, Faculty of Veterinary Medicine, Damanhour University, 22511 Damanhour, Egypt. 3These authors contributed equally: Manal T. Hussein and Abdelraheim H. Attaai. *email: [email protected]

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Calcium ions (Ca­ +2) are an important moderators, acting as major secondary messengers, of several vital physiological processes, including diferentiation and morphogenesis, membrane excitability and conductivity, axonal transport, synthesis and release of neuroactive substances and some neurotransmitters (phenomena of synaptic plasticity). Te calcium signal is mediated by a superfamily of structurally related proteins, generally known as calcium-binding proteins (CaBP), such as CB, PV, CR and S100 protein­ 7–11. Tese molecules pos- sess characteristic structures which can bind Ca­ +2 with high afnity­ 12–14. Altered CaBP expression may lead to pathological and neurodegenerative conditions, such as in patients sufering from epileptic seizures are lacking neuronal ­PV15–17. It is important to study the cerebella of diferent animals, in order to expand our knowledge about the cerebellum and many cerebella of diferent species have been studied. However, there is a paucity of information concerning the morphology of camel cerebellum, even in the Middle East and Asia, where camels are important in their economies. It will be interesting to explore the cerebellum of an animal such as the drom- edary, with very long legs and neck, who lives in a hot desert and walks long distances under hard conditions. All these factors may require specialized architecture in its cerebellum. Terefore, the present study was designed to achieve a detailed description of the morphology and cellular organization of the camel cerebellum. Because of the high importance of the calcium ions as a necessary moderator the current work also aimed to investigate the distribution of calcium binding proteins (CaBP) such as calbindin D-28K (CB), parvalbumin (PV) and calretinin (CR) in diferent cerebellar cells including the non-traditional neurons. Material and methods Collection of specimens. Te Ethics Committee of Alexandria University, Egypt approved this study. In this study camel’s were collected from ten clinically healthy mature camels (Camelus dromedarius) (4–6 years old) heads without sex diferentiation. Te animals were sacrifced in Alexandria slaughterhouse in Alexandria province, Egypt according to local ethical board guidelines of Egyptian slaughterhouses (Alexandria animal ethics committee 24122018). Afer slaughtering the camel, the entire head was separated from the neck, and saline was injected in the common carotid artery to wash the blood from the head circulation. Te 4% para- formaldehyde in PBS was injected in the common carotid artery for at least 30 min then the skull was opened to obtain the brain. Every brain was hemisected into two halves before immersed in the same fxative to allow more exposure to the fxative. Afer have been stored at 4° C for 3 days. Te cerebellum was carefully dissected from the brain and the weight of the brain and the cerebellum were weighed using sensitive balance.

Light microscopic study. Te tissue blocks were prepared for parafn cutting. Briefy, the dehydration of the fxed tissues was carried out using ascending grades of ethyl alcohol. Te samples were cleared by immers- ing in xylene then embedding in parafn wax (Sigma Aldrich, USA) according to the standard methodology described by ­Romeis18. Step serial sagittal Sects. (100 serial sections) from the vermal region of folia VI, VII, VIII were cut at 6–8 µm thickness using a Richert Leica RM 2125 Microtome, Germany, and mounted on glass slides. Some sections were stained with cresyl violet (to stain cell bodies) together with luxol fast blue (for myelinated fbers) and other section with Grimelius silver nitrate impregnation (for both cell bodies and processes)19.

Immunohistochemical staining. Immunohistochemical staining had been performed on the parafn blocks as mentioned before by Abdel-Maksoud, et al.20. Sections were deparafnized with xylene and hydrated with a descending grade of ethanol then washed with 0.1 M PBS (3 × 10 min). To decrease the masking of anti- gen epitopes, the antigen retrieval was carried out using 0.1 M sodium citrate bufer solution (pH = 6) for 7 min using a microwave (600 W). Ten, sections were cooled to room temperature for 20 min and washed with PBS (pH 7.4) for 10 min. Afer blocking the endogenous peroxidase activity with 3% H­ 2O2 in H­ 2O for 30 min at the room temperature (RT), the sections were washed with PBS (3 × 5 min), then the sections were blocked with 10% normal donkey serum (NDS) + 0.2% Triton-X100/PBS for 2 h at RT. Subsequently, sections were incubated overnight at 4 °C with the following antibodies: rabbit polyclonal anti-CB [1:200, Genemed Biotechnologies, cat- alogue NO. (61-0061)], rabbit polyclonal anti-PV [1:200, Termo Fisher Scientifc catalogue NO. (PA1-3945)] and mouse monoclonal anti-CR [1:200, Termo Fisher Scientifc; catalogue NO. (MA1-16629)]. Sections were rinsed 3 × 10 min in 0.2% Triton-X 100/PBS and incubated with biotinylated IgG goat anti-rabbit (catalogue NO.E043201-8) and IgG goat anti-mouse secondary antibody (catalogue NO.P0447) (from Dako, Hamburg, Germany) diluted at 1:200 for 2 h at RT, followed by incubation with Vectastain ABC (Avidin–Biotin complex) reagent for 45 min in a humid chamber at room temperature. Visualization of the reaction was carried out with 0.04% 3,3′-diaminobenzidine (DAB) and 0.003% H2O2 in 0.05 M Tris–HCl bufer (pH 7.5) for 5–10 min. Te sections were dehydrated in a graded series of ethanol, cleared with xylene and covered with DPX. Negative con- trol were performed by incubating sections without the primary antibodies. Immunohistochemical staining was evaluated by LeitzDialux 20 Microscope and photos were photographed by canon digital camera (Canon Power- shot A95) in the department of anatomy and histology, faculty of veterinary medicine, Assiut University, Egypt.

Morphometric and statistical analysis. Te morphometric studies were performed on both light and immunohistochemical images of the camel cerebellum using Image-J sofware. Te dimensions and counting of the cells were performed by measuring at least 10 sections from every cerebellum, and the results were presented as the mean of the measurements and considered as representative at these regions. Te cross sectional area (CSA) is the measured area of the cells containing the whole view of the nucleus. Te diameters are the longest and shortest diameter of the cells, containing the complete view of the nucleus, passing through the nucleus. Te cellular density per CSA was performed by counting cells in a defned area and were fnally calculated to 0.01 ­mm2. Te linear density for PC was performed by counting the PC in a defned length of 1 mm. We chose the linear density for PC because they appear in the sections arranged in a line between the granular and molecular

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CSA (μm2) Diameter (μm) Density (/0.001 ­mm3) Density per CSA (0.01 ­mm2) Granule cells 35.7 ± 2 7.1 × 6.2 1289 ± 148 80.6 ± 9.26 CSA (μm2) Diameter (μm) Density (/0.001 ­mm3) Linear density (/1 mm) Purkinje cells 797.73 ± 90 43.76 × 31 139.4 ± 12.8 7.4 ± 1.1

Table 1. Te dimensions and densities of the granule cells and Purkinje cells in the camel cerebellum. (CSA); cross sectional area.

Figure 1. Te anatomy of the camel cerebellum. (a) Te camel cerebellum lies caudal to the cerebral hemisphere and above the medulla oblongata. It consists of a central vermis which fanked by the cerebellar hemispheres. Its surface shows a lot grooves. (b) A sagittal view of the camel cerebellum shows the characteristic central which covered by the gray matter.

layer. To evaluate the ratio of immunopositive PC for diferent markers, we counted 100 PC manually from 3 diferent sections per animal. Some measurements for the size and density of PC and granular cells are sum- marized in Table 1 . Results Anatomical observation. Te camel cerebellum weighed 49.2 gm and the entire brain weighed 355 gm, therefore, the camel cerebellum averages 13.86% of the brain. Te anatomical features of the camel cerebellum were comparable to those of other mammals. Te cerebellum lies caudal to cerebral hemispheres. It consists of 2 symmetrical lateral halves, the 2 cerebellar hemispheres which fank the medial part, the vermis. Te dorsal surface of camel cerebellum has many fssures of variable depths subdividing the entire cerebellar surface into a considerable number of leaf-like lamellae (cerebellar folia) which separated by sulci (of variable depth) (Fig. 1a). Te internal branched white matter is covered by the foliated outer giving it the appearance of a tree, and hence its older name, the (Fig. 1b).

The microanatomy. W applied some histochemical staining, such as cresyl violet (cell bodies) together with luxol fast blue (myelinated fbers) and silver impregnation (for both cell bodies and processes) on the camel cerebellar tissues to visualize the cytoarchitecture of the diferent types of neurons, and axons,. Histo- logically, the architecture of the camel cerebellum is conserved as diferent mammals and consists of the medulla covered by 3 layered-cortex; made up of an outer molecular layer, middle ganglionic (Purkinje) cell layer and inner granular layer (Fig. 2a,b). Surprisingly, we noticed a wide region of white matter that reach distally to the pial surface, without covering with cortical tissue (Fig. 2b, supplementary fle). Te outer molecular layer consisted of relatively few neuronal cell bodies and many unmyelinated fbers (Fig. 2c). Polymorphic neuronal cells were observed in the molecular layer and the outer half showed more stel- late cells, than the basket cells found in the deeper half, (Fig. 2d). Notably, the cresyl violet did not show as many cells in the molecular layer compared to the silver stain. Moreover, diferent processes of numerous cells could be visualized by the silver stain (Fig. 2c,d). Te dendrites of the PCs appear to extend through the molecular layer (Fig. 3a). Te axons of basket cells make a meshwork around the cell bodies of PCs (Fig. 3a), while their dendrites extend perpendicular to the dendretic tree of PC (Fig. 3b). Te axons of granular cells traversing the molecular layer to variable depth then bifurcated at right angles to give rise to parallel fbers (Fig. 3c). Whereas, both silver and Luxol fast blue could not visualize the climbing fber nor the mossy fbers. Te PC layer made up of one row of cell bodies was located at the border between the molecular and granu- lar layers. Te PCs in camel were characterized by their large, fask-shaped cell bodies. Each cell contained a vesicular nucleus with a prominent circular nucleolus (Fig. 3a,c) and we observed some Nissles granules arranged

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Figure 2. Histochemical staining showing the general architecture of the camel cerebellum. (a) Cresyl violet with luxol fast blue, (b) silver staining showing the cerebellar medulla white matter (W) and three layers of the cerebellar cortex; M (molecular layer), P (Purkinje layer) and G (Granular layer). (b) Notably, a wide region of white matter that reach distally to the pial surface, without cortical tissue covering (arrowheads). (c) Te outer molecular layer (M) consisted of relatively few numbers of neuronal cell bodies (arrowheads). (d) Silver staining showing more polymorphic neuronal cells in the molecular layer compared to cersyl violet staining. Moreover, diferent processes of numerous cells could be visualized by the silver stain (arrows).

peripherally at certain focuses (Fig. 3d). Silver staining showed various processes synapsing with the cell bodies of the PCs. Te PCs dimension measured 43.76 × 31 µm, with a CSA (cross sectional area) of 797.73 ± 90 µm2. PC linear density was 7.4 ± 1.1 cells/1 mm. Te calculated density was 139.4 ± 12.8 cells/0.001 ­mm3 volume. Some of the measurements for the size and density of PC and granular cells, using ImagJ sofware, are summarized in Table 1 . Te inner most granular layer is extremely cellular and densely populated by small spheroid somata with dark-stained nuclei and scanty cytoplasm (Fig. 4a,b). Te granular cells arranged in clumps, rosettes or cords, and many spaces exist within the granular layer, most of them are lightly stained with silver stain (Fig. 4b,c). Te measured 7.1 × 6.2 µm with CSA 35.7 ± 2 µm2. Granule cell density was 80.6 ± 9.26 cells/0.01 mm­ 2 CSA. Te calculated density was 1289 ± 148 cells/0.001 mm­ 3 volume. Luxol fast blue staining showed that white matter and many myelinated axons traverse through the granular layer from the layer and the molecular layer either individually or in bundles (Fig. 4a,b). Addition- ally, many luxol fast blue stained endings surround the somata of the PCs, which are likely from axons of basket cells (Fig. 4b). Occasionally, some bundles run immediately beneath and parallel to the arranged PC somata (Fig. 4d,e), which are likely axons from Lugaro cells, basket cells as well as collaterals from PC axons. Moreover, we were able to detect non-traditional large neurons, such as Lugaro (Fig. 4e) and synaromatic neurons (Fig. 4f), with cresyl violet and silver staining.

Molecular characterization of the CaBP in camel cerebellum. Immunohistochemical staining against diferent molecules of the CaBP in camel cerebella such as CB, PV and CR were performed. However, immunopositive structures were detected throughout the cerebellar cortex, with layer-specifc patterns of expression. Te immunoreactivity was visualized in neuronal bodies, and in some instances, in dendrites and axons. Negative control sections incubated without the primary antibodies revealed no staining for neurons or fbers (See supplementary fle).

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Figure 3. Histochemical staining showing the camel cerebellar cortex. (a) Silver staining showing the dendritic arborization (D) of the Purkinje cells which extend through the molecular layer. Te outer half of the molecular layer contains stellate cells (S), and basket cells (B). Te axons of basket cells make a meshwork around the cell bodies of Purkinje cells (arrows). Te Purkinje cells (PC) in camel were characterized by their large, fask- shaped cell bodies. Each cell contained a vesicular nucleus (N) with a prominent circular nucleolus. (b) Basket cell (B) extends perpendicular to the dendretic tree of PC. (c) Silver staining showing the parallel fbers (arrows). (d) Cresyl violet staining showing the Nissl’s granules (arrowheads) which arranged peripherally in the Purkinje cells at certain focuses.

Calbindin‑D 28 k (CB) expression in the camel cerebellum. Te Purkinje neurons showed the high- est CB immunoreactivity, which demarcates the characteristic morphology of the Purkinje neurons throughout its extent (Fig. 5a). Moreover, the dendrites extend the whole length of the molecular layer and showing obvious dendritic arborization including the primary and secondary trunks and the spiny branchlets (Fig. 5a). However, seldom low CB-immunopositive PC bodies were observed (Fig. 5b). Te ratio of CB-immunopositive cells were about 99 percent of the PC. Te nature of the strong immunoreactivity was seen in densely packed homogeneous deposits in their cytoplasm, and in some cases, a similar immunoreactivity was seen within the nuclei (Fig. 5c,d). Strong CB-ir fbers were seen beneath and surrounding the PC, which are likely from basket and/or Lugaro cells (Fig. 5c). Also, some coarse CB-immunopositive fbers were traversing through the granular layer and were ori- ented obliquely or vertically till reaching the white matter. Tey are characterized by varicosities along with their profle and some of them could be traced originated from PC (Fig. 5d). Interestingly, the immunoreactivity was

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Figure 4. Histochemical staining showing the camel cerebellum. (a, b) Cresyl violet staining showing the granular layer cell layer which is extremely cellular and populated by small spheroid bodies. Luxol fast blue staining showing the white matter (W) and many myelinated axons (Ax) traverse through the granular either individually or in bundles. (b) Axons of basket cell surround the Purkinje cells (arrowheads). c: Silver staining showing the granular cells arranged in, rosettes (circles) or cords (irregular shapes). (d, e) Bundles run just beneath and parallel to the arranged Purkinje cell bodies (arrows). Arrowheads show the glomerular islands. (e, f) Non-traditional large neurons, such as Lugaro (L) and synaromatic neurons (S) with cresyl violet and silver stains respectively.

observed in the spiny branchlets with many irregularities on their surface indicating dendritic spines (Fig. 5e). Tere are also abundant immunopositive punctate elements (puncta) distributed in the neuropil between neu- rons of the molecular layer (Fig. 5e). All of the abovementioned immunopositive structures impart an intense coloration to the. In the molecular layer, CB-ir cells showed various degrees of intensity. Te immunoreactivity was observed within the cytoplasm of the bodies and extended into the initial segments of the processes. Basket cells were rec- ognized by their shape, orientation and position as well as their elongated somata whose long axes were parallel

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Figure 5. Calbindin-D28k (CB) immunoreactivity in camel cerebellum. (a) CB immunoreactivity was obvious in almost all cerebellar cortical layers and the white matter (W). Purkinje cells (PC) showed the highest CB immunoreactivity. In addition, the dendrites extend the whole length of the molecular layer, showing an obvious dendritic arborization (D). (b) Seldom low CB-immunopositive PC bodies were observed (arrow). of Lugaro (L) and candelabrum neurons (CN) were observed. (c) CB immunoreactivity was seen as densely packed homogeneous deposits in the cytoplasm of the Purkinje cells and within the nuclei (N). Notice, CB immunoreactive fbers beneath and surrounding the PC, which are likely from basket and/or Lugaro cells (arrow). Immunoreactivity was observed in the spiny branchlets (arrowhead). (d) CB-immunopositive fbers (arrow) were traversing through the granular layer (G) and were oriented vertically till reaching the white matter. Inset showing the varicosities (arrowheads) along the axon profle which originated from the PC. (e) CB immunoreactivity was observed along the dendritic arborization till reaching the pial surface (P), arrowheads indicates the spiny branchlets of the dendrities.

to the PC layer and localized in the deeper region of the molecular layer (Fig. 6a). Notably, there was CB-ir ring surrounding the somata of the PC, which originated from the axon terminals of the basket cells (Fig. 6b). Te stellate neurons were characterized by their rounded/polymorphic somata and their settlement in the outer

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zone of the molecular layer. Te dense CB-ir was also observed within the cytoplasm of PC dendritic tree which extend along the distal ramifcations, just underneath the pial surface (Fig. 6c). In the granular layer, moderate CB-ir was observed in a fraction of the granular cells, however, the expression was heterogeneous. Te immunopositive cells displayed moderate, fnely granular cytoplasmic immunoreactivity (Fig. 6d,e). Te large Golgi neurons showed positive CB-ir (Fig. 6d). Tere were islands of CB-immunopositive a cellular spots in the granular layer among granule cells (islands of Held) (Fig. 6d,e). Te heterogeneous CB expression was also obvious in the medulla, where fbers reacted diferently with CB. Moreover, the perivascular large non-traditional neurons showed positive CB-ir (Fig. 6f).

Parvalbumin (PV) expression in the camel cerebellum. PV-ir was obvious in the PCs and molecular layer cells (Fig. 7a). Parvalbumin was expressed by the PCs which appeared as a difuse granular reaction within the cytoplasm of the cell bodies and the dendrites, whereas, the nuclei showed less immunoreactivity. Notably, PV-ir was less intense compared to CB (Fig. 7b) and the ratio of PV-immunopositive cells were about 92 percent of the PC. Molecular layer cells showed higher PV-ir compared to CB. Moreover, the molecular layer cells were heterogeneously expressing PV. Its expression was also difused within the cells with strong expression by the nuclear membrane. Te processes of basket cells could be visualized clearly using this marker. Tese processes extending from basket cells towards the PCs and form a network surrounding the PC bodies (Fig. 7c,d). Te stellate cells were expressing PV, with varying degrees of expression in their nuclei. In contrast to PC and basket cells, we could not detect the processes of stellate cells (Fig. 8a,b). We could detect solitary fbers traversing the granular layer, which are likely the axons of PCs (Fig. 8c). Furthermore, some, but not all, fbers of the white matter were expressing PV (Fig. 8d).

Calretinin (CR) expression in the camel cerebellum. CR-ir was obvious in almost all cell layers with varying degrees (Fig. 9a). Te molecular layer cells showed strong immunoreactivity for CR (Fig. 9a). Te den- sity of CR immune positive cells of the molecular layer was 129 cells per 50,000 μm3. Te basket neurons were recognized by their elongated body whose long axis parallel to the cerebellar surface and localized in the inner region of the molecular layer (Fig. 9 b–d). In addition to the well-known arrangement of the basket cell fbers surrounding the PC somata, we noticed also the dendrites of the basket cells form synapses with dendrites of the Purkinje cells (Fig. 9c,d). Te stellate neurons were characterized by their polymorphic body and their localiza- tion in the outer zone of the molecular layer (Fig. 9e). PC showed any or very weak CR-ir (Fig. 9c,d) with a ratio of CR-immunopositive cells were about 4 percent of the PC. Te granular layer showed positive CR-ir and is composed of two main groups of neurons: the granule neurons (granules) and the large neurons (Fig. 10a). Te granule cells have a small, spheroid body and they aggregated in cords or clumps leaving large spaces in between. Large neurons showed stronger CR-ir and have voluminous polygonal or ovoid body. Tey involve the neuron of Golgi, one of the fve traditional corticocerebellar neurons (Fig. 10b). Besides, several other large neuron types, generically indicated as non-traditional neurons were demonstrated. Te expression of diferent CaBP in difer- ent cerebellar cell components are summarized in Table 2.

Non‑traditional large neurons. Neurons of Lugaro (NL or horizontal neurons). Tey are located in the external zone of the granular layer and in more/less close contact with PC layer. Teir bodies are fusiform and horizontally oriented, parallel to the surface of the folium. Te processes extend from the 2 poles of the body and are oriented horizontally along the boundary between the granular layer and PC and forms synapses with PC. Tey showed positive immunoreactivity for CB and CR (Fig. 5b and Fig. 10c).

Synarmotic neurons (SNs). Tey are located in the inner zone of the granular layer near the subcortical white matter. Teir bodies are ovoid and horizontally oriented. Tey showed positive reactivity with silver impregna- tion, CB and CR (Figs. 4f, 6e and 10d).

Candelabrum neurons (CN; also known as an intercalated neurons). Tese neurons have a vertical major axis and pear-shaped bodies. Teir bodies are squeezed against the bodies of Purkinje neurons. Tese neurons showed positive immunoreactivity for CB and CR (Figs. 5b and 10e).

Unipolar brush neurons (UBN; monodendritic neurons). Tese neurons have round, or ovoid and vertical cell bodies. Tey are localized throughout the granular layer. A single thick dendrite trunk originates from the exter- nal body pole, extends for a short distance and gives rise to a push-like end. Tese neurons could be visulaized only by its positive CR-ir (Fig. 10 f–h). Discussion Te current study aimed to extend the knowledge about the camel cerebellum that helps to learn more about the cerebellum. We demonstrated the detailed characterization of the architecture, both of cellular and fber components, of the cerebellar cortex in the camel cerebellum. To this end, diferent histochemical stains were applied. Furthermore, numerous markers were used to visualize the diferent CaBPs by immunohistochemical analysis. One of the characteristic features in the results of histological stains of the camel cerebellum was that the granular cells were arranged in cords or ­rosettes21,22. According to our observation the cells in the granular layer were not crowded and many spaces were observed. Te relative weight of camel’s cerebellum is 13.86% which is larger than that of donkey 10.8%23. When com- pared to granule cell of other animals, those of camel were the smallest, CSA 35.7 ± 2 µm2, where it was 49.7, 50.7,

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Figure 6. Calbindin-D28k (CB) immunoreactivity in camel cerebellum. (a) Basket neuron (arrow) was recognized by their elongated body whose long axis (arrowheads) parallel to the cerebellar surface. (b) CB immunopositive rim surrounding the cell body surface of the Purkinje neurons (PC), which corresponding to axon terminals of the basket neurons (arrows). (c) Te outer zone of the molecular layer showing the polymorphic stellate cells (S). Notice, the strong CB immunoreactivity was also observed within the molecular layer coming from the deeply stained dendrites of PC (arrows) which reaches to the distal ramifcations just beneath the pial surface (P). (d, e) Te granular cell layer showing a moderate CB immunoreactivity, however, the expression was heterogeneous. (d) An inset showing the Calbindin immunoreactivity by Golgi neurons. (e) Synaromatic (SN) large neurons were observed among the granular cell layer. A cellular spots or island in the granular layer (arrowheads) were observed. (f) Te perivascular large non-traditional neurons (arrowheads) surrounding the blood vessels (BVs) showed positive immunoreactivity with CB in the cerebellar medulla or white matter (W).

52.9, 63.8 and 68 µm2 in humans, leopards, chimpanzees, tigers and Girafes, respectively­ 24. Granule cell density was 1289 ± 148 cells/0.001 mm­ 3, which was more than that of elephants and Bottle-nose dolphin (807 ± 76 and 1083 ± 112 cells/0.001 ­mm3, respectively). It was comparable to that of sheep and common porpoise (1205 ± 131

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Figure 7. Parvalbumin (PV) immunoreactivity in camel cerebellum. (a) Parvalbumin immunoreactivity was observed in the Purkinje cells (PC) and in the stellate (S) and basket cells (B) of the molecular layer cells (m). (b) PV was expressed by the Purkinje cells which appeared as a difuse granular reaction within the cytoplasm of the cell bodies and the dendrites. Notably, the expression was less intense compared to CB. (c, d) Te processes of basket cells (B) could be visualized using this marker. Te processes (arrowheads) extend from basket cells towards the Purkinje cells and form a network (arrows) surrounding the Purkinje cell bodies. P pial surface, G granule cells.

and 1254 ± 116, respectively). Te density was lesser than that of bull, horse and man (1416 ± 141, 1503 ± 165 and 1609 ± 171 cells/0.001 ­mm3, respectively). Te density was far lesser than that of rat, mouse and squirrel monkey (3216 ± 287, 3111 ± 341 and 2990 ± 301 cells/0.001 ­mm3, respectively). Tese results run in parallel to notion that the cell density (Number of cells) decreases with higher brain weight ­animals25. PC dimension in camel measured 43.76 × 31 µm, with a CSA of 797.73 ± 90 µm2. It is larger than that of rat (21 × 25 µm), mice (CSA 188 µm2) and cat (width 29 µm), whereas, it is comparable to that of man (30–35 × 50–70) (reviewed ­in26). Tere three diferent methods to analyze the PC density; to be PC number with respect to volume in µm3, to area in µm2 or in certain length (linear). We preferred the later because it is the simplest and not subjected to calculations; however, we presented all three values. Due to the ambiguity inherent in measuring the volume of the PC layer we took the PC density to be number with respect to the area of the PC layer interface rather than to a volume. Te units were accordingly neurons/mm227. PC linear density was 7.4 ± 1.1 cells/1 mm and the calculated density was 140.8 ± 2 cells/1 mm­ 2 or 139.4 ± 12.8 cells/0.001 mm­ 3 volume. We thought that the PC densities to be high because of the large body and long extrimites of camel; however, they had the lowest densities among several previously studied species. PC linear density in camel was much less than that of mice (38.5 ± 1.2 cells/mm)28. Te calculated PC density in camel was also much less than that of rat (930 cells per mm­ 2 in the posterior lobe), cat (567 per mm­ 2) and monkey (510 cells per mm­ 2). PC density in camel was the nearest to that of man (300 cells per ­mm2) among diferent species; however, it still far less than that of man, about half the densities (reviewed ­in26). Tese low PC and granule cell densities were recorded also in the African ­elephant29. Together with the abundant cerebellar islands, this might indicate an abundant synapses in the neuropil to control the complex motor patterns and to process the sophisticated neural information which increases during ­phylogenesis29. Te CaBPs are widely expressed throughout the nervous system however they are among the most abundant proteins in the cerebellum­ 8. Tese proteins expressed in the peripheral tissues including the expression of CR in the thymus, CB in the kidney and PV in the skeletal muscles and endocrine ­tissues15,30. In fact, no data on the expression of the CaBP within the camel cerebellum are available to date. Terefore, the results of the current study provide for the frst time the detailed characterization of cellular, together with fber, components in the

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Figure 8. Parvalbumin (PV) immunoreactivity in camel cerebellum. (a, b) Stellate cells in the upper molecular layer show positive immunoreactivity for PV. (c) Te axons of Purkinje cells fbers (arrow) traversing the granular layer. (d) some fbers of the white matter were expressing PV.

camel cerebellar cortex. To this end, diferent immunohistochemical analyses were applied using markers for CB, CR and PV. We observed that the expression of each molecule was diferentially expressed among diferent cerebellar populations. As a ubiquitous second messenger, ­Ca2+ has been shown to regulate membrane ­excitability31, dendrite ­development32, ­synaptogenesis33, and many other processes contributing to the neuronal primary functions of the information processing and memory storage­ 34. CB was detected in the nervous system­ 35, afer its discovery in the chicken intestine­ 36. Its expression is started early in cerebellar development in fetuses and newborn kittens during neuronal migration and is afected in aged ­dogs37. CB expression was intensely and widely distributed in diferent compartments; neuronal bodies, their main processes and the axon and dendritic terminal compartments, thus indicating its importance for normal cerebellar function. Tis some studies of various mammalian species stated that CB expression was predominant in Purkinje ­neurons38,39, the present study demonstrated that the CB was expressed in almost all cortical neuron types, however, with diferent intensity. Te PCs showed the highest CB immunoreactivity, which distributed throughout the cytoplasm of cell bodies, axons and dendritic tree. Terefore, CB demarcates the characteristic morphology of the Purkinje neurons throughout its extent. However, a few low CB-immunopositive PC bodies were observed. Te appearance of the detailed morphology of PC, including its very distal tiny branchlets and spines, using these antibodies, made it a marker of choice to study PCs­ 40. Te cytoplasmic abundance of CB is consistent with the previous studies that demonstrated the interaction between CB and cytoplasm proteins, such as myoinositol mono-phosphatase and protein M­ 41. Additionally, the nuclear localization of CB in the PC supports the hypothesis that is plays a role in the regulation of gene ­expressions42. Tis homogeneous, intense expression of CB along the entire cytoplasm of the PC indicates the involvement of CB in many neuronal func- tions, especially in this large neurons which receive huge numbers of synapses from numerous types of cells. Furthermore, the experimental data demonstrated that CB plays a neuroprotective role against the oxidative stress or toxic efect of prolonged stimulation by excitatory amino acids (i.e.: glutamate; aspartate)43. Terefore, the absence or alteration in the expression of calcium-bufer proteins by any way will result in marked abnormalities in cell fring, with alterations in simple and complex spikes­ 37. Tis will be accompanied by many neurological disorders and neurodegenerative conditions concerning motor coordination and sensory ­functions44. It has been reported that PC loss CB-ir in some viral encephalitides from HIV encephalitis in ­humans45, rabies-infected ­cattle37, neonatal Borna disease infection in rats­ 46, and experimental infection of in ­mice47, or aging in ­dogs48. Tese notions postulate a relationship between loss of CB-ir in PC, altered calcium homeostasis and calcium

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Figure 9. Calretinin (CR) immunoreactivity in camel cerebellum. (a) Calretinin immunoreactivity was obvious in almost all cell layers with varying degrees. (b) Te molecular layer cells (M) showed strong immunoreactivity for calretinin. (c, d) Te basket neurons (B) were recognized by their elongated body whose long axis (arrowhead) parallel to the cerebellar surface and localized in the inner region of the molecular layer. Purkinje cells (PC) showed any or very weak immunoreactivity for Calretinin. (e): Te stellate neurons (S) were characterized by their polymorphic body and their localization in the outer zone of the molecular layer. Mol molecular layer, BV blood vessel.

internal bufering and dysfunctions in GABAergic neurotransmission. Te current study supplies surprising fndings of the expression of CB within diferent neuronal populations such as the non-traditional large neurons (lugaro and synaromatic neurons) and granule cells in the granular layer and other inhibitory GABAergic neurons (basket and stellate cells) in the molecular layer of the camel cerebellar cortex. Our results are in line with that observed the positive immunoreactivity for CB-D28k in the bovine GABAergic inhibitory interneurons such as the basket cells, Golgi and Lugaro neurons , elephant­ 25 sheep­ 49. Te positive immunoreactivity for CB was also detected within the stellate and basket cells of the human cerebellar cortex in the molecular layer. Recently, the immunoreactivity was observed in the granule neurons, and in the non-traditional large neurons such as the candelabrum, ellpoisidal, Lugaro, and the synaromotic neurons­ 42. Terefore, we concluded that CB in the camel cerebellum plays a primary role in the regulation of cerebellar cortex functions, in addition to, its main role in integrative functions of the cerebellar cortex, which involoves sensorimotor and cognitive ­functions42. CB Purkinje neurons are known as the main source of projective axons and the sole output from the cerebellar cortex. SNs have recently been reported as the second type of projective neurons in the cerebellar cortex which showed positive CB-ir50. Te expression of CB was linked to the GABAergic projective neurons (Purkinje and SN), and we assumed that CB is mainly associated with the neurotransmission of inhibitory type­ 42. In line with this notion, the current study demonstrated the expression of CB in the GABAergic inhibitory neurons including stellate, basket, and Lugaro neurons­ 51,52.On the other hand, we found also that CB expression was also demon- strated in some glutamatergic excitatory neuron including a large subpopulation of the granule neurons. Taken together, our results indicate the expression of CB by neurons is implicated in both the projective (extrinsic) and regulatory (intrinsic) circuits of the camel cerebellar cortex and there is no correlation between CB expression and type of neurotransmitter­ 42,52. PV was expressed by PC, however, its intensity in the and dendrites was lower when compared to CB. Moreover, PV showed variations in the staining intensity within the PC cells. Te same results were demon- strated in the cerebellum of rat, primates and human­ 8,35. Two subpopulations within the molecular layer were also expressing PV, the stellate and basket ­cells53. Tese inhibitory GABAergic interneurons receive excitatory inputs from the granule cells and climbing fbers and exert inhibitory signals to the ­PC54. Both populations have relatively comparable morphologies with short dendritic trees, which are organized in an almost right angle

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Figure 10. Calretinin (CR) immunoreactivity in camel cerebellum (posterior lobes). (a) Te granular layer showed moderate positive immunoreactivity for CR in the granule neurons (G) and in the Golgi and other large non-traditional neurons. (b) Golgi (Go) neurons showed a voluminous polygonal or ovoid body. (c) Neurons of Lugaro (L) showed fusiform and horizontally orientated body. (d) Candelabrum neuron (CN) showed pear- shaped body. (e) Synaromatic neuron (SN) showed ovoid and horizontally orientated body. (f–h): Unipolar brush cells (UB) have round/ovoid, vertical cell bodies projecting a single thick process (arrow) that have push- like end (arrowhead).

to the dendrites of the PC cells. Te axons of basket cells extend and, as their name implies, organized around the PC somata as a basket­ 47 Te dendrites of both cells and the axons of the stellate cells form synapses on the dendritic tree of Purkinje cells. In contrast to the human cerebellum, we found that PV was not expressed by the GABAergic Golgi cells within the granular layer of the camel cerebellum­ 54. Although the PCs have both CB and PV, the autism spectrum disorder (ASD) was linked to PV reduction. Te cerebellum neuropathology in ASD was frst detected over 35 years ­ago55, where the signifcant reduction in PV-expressing PC in autistic individuals is considered a histopathological feature of ­autism56. Terefore, altered calcium metabolism plays a key role in ASD pathophysiology, where it might, in turn, impact GABAergic signaling­ 57. PV expressing neurons are more subjected to degeneration. In contrast, CR and/or CB expressing neurons are more likely resistant reviewed ­in58. CR is structurally related to CB and was frst described in 1987 in embryonic chick retina, hence its name is driven from CB and retina­ 59,60. According to the speed of Ca­ 2+ bufering, CB is the fastest, PV is the slowest

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CB (fast) PV (low) CR (moderate) Purkinje (Soma) + + + + + + Axon + + + + − Dendrites + + + + + − Nucleus + + + + + + Stellate (Soma) + + + + + + + Processes − − + Basket (Soma) + + + + + + + Processes + + + + + + + Granule (Soma) + − + + Golgi (Soma) + − + + + Lugaro (Soma) + + − + Processes + + + UBC (Soma) − − + + + Processes + + Synaromatic (Soma) + − + + + Candelabrum (Soma) + − + + +

Table 2. Intensity of staining in diferent cerebellar cells.

and CR is sharing some kinetic properties of both fast and slow bufers in modifying ­Ca2+ transients due to the cooperative ­Ca2+ binding of ­CR59–61. In the camel cerebellum, the CR is expressed in almost all cells except the PC which showed any or very weak CR-ir. However, recently, it has been reported that there is CR-ir in the migrating PC from the ventricular neuroepithelium in the developing human fetal ­cerebellum62, which suggest a possible role during migration. Tis observation was in line with Álvarez et al., 2008 who demonstrated the variability for calretinin expression in the purkinje cells of the sheep. Te authors claimed the great variability in CR-immunopositivity cannot be associated to methodological variables instead, this notion confrms the idea of a heterogeneous functional organization for the cerebellar cortex within an apparent homogeneous anatomical and histological structure. In camels, Te majority of granule cells were expressing CR, however, was relatively weaker than other cells. Tis was in line with that reported in rodents­ 63–65. Moreover, CR-ir was detected also in the axons of granule cells, the parallel fbers, suggesting an important role in ­Ca+2-dependent synaptic ­plasticity65,66. CR was also expressed by large non-traditional neurons of the granular layer: unipolar brush cells (UBC), Lugaro cells, synaromatic cells and candelabrum cells. Tese populations were more intensely stained with CR antibody than the surrounding granule cells, supporting the notion that they contain higher concentrations of CR. Schwaller, et al.54 and colleagues reported only that CR is expressed by unipolar brush cells (UBC)67 and Lugaro cells in ­rodents68. Te authors did not mention whether other large non-traditional neurons expressed CR.68. Lugaro cells and their processes are usually located just below the PC layer, which we could detect CB and even using the Luxol fast blue staining. Tey are believed to exert an indirect inhibitory infuence on PC via a glycinergic inhibition of Golgi ­cells69. CR-ir by Lugaro cells is not conserved in all vertebrates, because Lugaro cells of Pigeon are not expressing it, instead they express secretagogin­ 70. We found UBC in the granule cell layer of the posterior cerebellar lobe in camel, similar to that described in rodents­ 65 and ­sheep71. Tey have a single, thick process which forms a bush-like tip which is reported to function as excitatory glutamatergic neurons­ 67. We found distinct large CR-ir globular cells deep in the granular layers suggestive to be Golgi cells. However, it has been reported that Golgi cells in all species except for guinea pig are immunonegative for ­calretinin72. It has been suggested recently that these cells might be a subtype of Lugaro cells in pigeon, where there are both globular and fusiform Lugaro cells­ 70. Tese results might be indicate that CR expression is not conserved in certain cells among all vertebrates. Te neuroprotective role of CR against calcium-stimulated cytotoxicity has been suggested­ 60. Because of the crucial role played by ­Ca+2 in neuronal physiology, it is not surprising that even modest impairments of ­Ca+2 homeo- stasis result in profound functional alterations. Despite the heterogeneous etiology of neurodegenerative disorders, as well as the physiological aging process, are all characterized by disruption of Ca­ +2 homeostasis and signaling. Conclusion In summary, the results of the current study achieved a complete map for the neurochemical organization of CaBP expression and distribution by diferent populations in the camel cerebellum. Understanding of these data under normal conditions represents a prerequisite for studying the cerebellar pathophysiology. PCs are expressing high CB, low CR and moderate PV. Molecular layer cells share the same expression profle; low CB and high CR and PV. Granule cells are expressing heterogeneous (low/moderate) CB, moderate CR and no PV, whereas this phenotype is the reverse in Lugaro cells. Golgi, UBC and synaromatic share the high CR-ir and no PV-ir and low CB-ir, however, UBC were not expressing CB.

Received: 15 June 2020; Accepted: 12 November 2020

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Competing interests Te authors declare no competing interests. Additional information Supplementary information is available for this paper at https​://doi.org/10.1038/s4159​8-020-78192​-7. Correspondence and requests for materials should be addressed to F.M.A. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afliations.

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